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Research


The work of the research groups led by Dr. John Quinn and Dr. John McGrath is focused on the microbial metabolism of phosphorus.  Specifically our research seeks to dissect the biochemistry and genetics of environmental phosphorus cycling in two main areas:

1.  Polyphosphate metabolism in environmental microorganisms.

Phosphorus (P) is an essential macronutrient for all organisms and plays some part in almost all life processes.  Yet despite this well-accepted central metabolic role, inorganic phosphate, in excess, represents a potentially serious environmental and ecological problem.  In particular, the enrichment of water bodies with phosphate makes an important contribution to the process of eutrophication which has developed into a serious water management problem throughout the world.  Freshwater lakes are considered to be eutrophic if the phosphorus concentration exceeds 35 g P/l; by this standard both Lough Neagh and Lough Erne can be considered to be highly eutrophic.  Indeed eutrophication poses the most widespread single threat to good water quality in Northern Ireland while in the United Kingdom 23% of lakes are considered to be eutrophic.  In most freshwater systems the limiting nutrient for algal and macrophytic growth is inorganic phosphate; current trends show that in the last 10 years the phosphorus inputs into both Lough Neagh and Lough Erne have inreased by more than 50%.  The excessive growth of algae or higher plants that occurs as a consequence of eutrophication leads to many water quality problems including oxygen depletion, increased water purification costs, a decline in the amenity and conservation value of waters, loss of livestock and the possible sub-lethal effects of algal toxins on humans using eutrophic water supplies for drinking (McGrath and Quinn, 2003, 2004).

Phosphate (P) removal from wastewaters is important for the control of eutrophication and is enforced by increasingly stringent legislation e.g. the European Urban Wastewater Treatment Directive 91/271 and 98/15.  P removal is currently achieved largely by chemical precipitation, although a biological process is regarded as preferable since chemical addition is expensive and results in the generation of large excess volumes of sludge (up to 20%).  Such a process would necessarily involve the 'luxury' uptake of soluble P by activated sludge microorganisms and its storage as intracellular inorganic polyphosphate (polyP) (McGrath and Quinn, 2003).

PolyP consists of a linear chain of P residues linked together by high energy phosphoanhydride bonds and is one of the most widely distributed natural biopolymers, having been detected in all organisms studied (Figure 1).  It is particularly significant within microorganisms where it may amount to as much as 10-20% of the cellular dry weight.  Despite its apparent microbial ubiquity, the exact physiological function of polyP remains unknown.  Suggested biological roles include that of a reservoir of energy and phosphate, a chelator of metals, a capsule material and a 'channelling' agent in the phenomenon of bacterial transformation.  It has also been hypothesised to play an important role in the response of microbial cells to nutritional and environmental stresses.

 

Our research focuses on the microbial metabolism of polyP.  In particular this has been to identify environmental conditions under which the ability to accumulate intracellular polyP is necessary for microbial survival (or at least confers a competitive advantage on cells that possess it).  Knowledge of such conditions might then be exploited to provide alternative and possibly superior treatment options for biological P removal.  This has led to the discovery of a hitherto unrecognised 'trigger' which induces polyP accumulation in environmental microorganisms.  Certain microbial isolates when grown at pH 5.0 - 6.5 rather than 7.5, display 3 to 4-fold enhanced levels of P uptake.  Increased P removal is accompanied by a 2 - 10.5-fold concomitant elevation in intracellular polyP content; this is visualised as intracellular polyP inclusions (Figure 2).  It has also been possible, in the laboratory using a synthetic wastewater medium, to increase the level of P removal by the microflora of a local conventional activated sludge plant - under fully-aerobic conditions - by more than 50% if the operational pH is adjusted to a value within the range 5.5 to 6.5, as opposed to the values between approximately 7.2 and 7.7 which are typical of current treatment practice (McGrath et al., 2001).  The biochemistry behind 'acid stimulated' polyP biosynthesis has been further investigated by us in two environmental isolates -Candida humicola and Burkholderia cepacia. (McGrath and Quinn, 2000; Mullan et al. 2002a; Mullan et al. 2002b) - and in various clinical isolates from cystic fibrosis patients (Tunney et al., 2006).

These laboratory results suggested that 'acid-stimulated' P uptake and polyP accumulation might be exploitable as a novel alternative technology for biological P removal.  Subsequently, in association with the Northern Ireland Water Service, Severn Trent Water Ltd, Extract Solutions Ltd. and Yorkshire Water Ltd. the potential of this system was investigated at pilot plant scale (Figure 3a).  A 2000 litre facility was constructed at New Holland Sewage Treatment Works (Belfast, Northern Ireland) to treat primary settled sewage.  This demonstrated that a decrease in operational pH from 7.0 to 6.1 resulted in a doubling of levels of P removal during the activated sludge process (Figure 3b).  Typically, this led to more than 80% total P removal from primary settled sewage (as legislated in Directive 91/271 and 98/15); pH reduction to 6.1 had no deleterious effects on other treatment parameters (Mullan et al., 2006).  The acid process therefore appears to represent an economical and viable alternative for P removal from wastewaters; a full-scale trial is now being initiated by the Northern Ireland Water Service, Wastewater Controls Ltd. and QUESTOR Technologies Ltd.

P removal from pilot plant

The full potential of this technology may however only be achieved if there is a firm understanding of its scientific basis.  Future research will therefore be directed at dissecting the underlying biochemistry and genetics of the acid stimulated phosphate uptake phenomenon.  Additionally our research will continue to focus on microbial polyphosphate metabolism.  For example, in association with researchers at University College Dublin, we are investigating the interaction between polyphosphate and the accumulation of polyhydroxyalkanoate - a carbon storage polymer used in the production of biodegradable plastic (Tobin et al., 2007).

These studies will continue to involve collaboration with local industry and fellow researchers at CEH-Oxford (Dr. A.S. Whitely), University College Cork (Dr. A. Dobson and Dr. J. Marchesi) and Dr Kevin O'Connor (University College Dublin).  To date this research has been funded by the EPSRC, EPA, Royal Society, BBSRC, QUESTOR Centre, Northern Ireland Water Service, Severn Trent Water Ltd, Yorkshire Water Ltd., Extract Solutions Ltd., Wastewater Controls Ltd, a Strategic Research Infrastructure (SRIF) grant for Environmental Engineering and Biotechnology, and Invest NI.

1.  Tunney, M., Moriarty, T., Mullan, A., McGrath, J.W., Quinn, J.P. and Elborn, J. (2006). Effect of reduced pH on inorganic polyphosphate accumulation by Burkholderia cepacia complex isolates.  Letters in Applied Microbiology, 42: 617 - 623.

2.  Mullan, A., McGrath, J.W., Adamson, T, Irvine, S. & Quinn, J.P. (2005) The application of low pH-inducible polyphosphate accumulation by environmental microorganisms to biological phosphate removal from wastewaters: a field study.  Environmental Science and Technology, 40: 296-301.

3.  McGrath, J.W. & Quinn, J.P. (2004). Biological Phosphorus Removal. In Phosphorus in Environmental Technology pp. 272-286 Ed Valsami-Jones, E. IWA Publishing.

4.  McGrath, J.W. & Quinn, J.P. (2003). Microbial Phosphate Removal and Polyphosphate Production from Wastewaters.  Advances in Applied Microbiology, Vol 52: 75-100.

5.  Mullan, A., Quinn, J.P. and McGrath, J.W. (2002). A non-radioactive method for the assay of polyphosphate kinase activity and its application in the study of polyphosphate metabolism in Burkholderia cepacia.  Analytical Biochemistry, 308: 294-299.

6.  Mullan, A., Quinn, J.P. and McGrath, J.W. (2002). Enhanced phosphate uptake and polyphosphate accumulation in Burkholderia cepacia grown under low pH conditions.  Microbial Ecology, 44(1): 69-77.

7.  McGrath, J.W., Cleary S, Mullan, A., & Quinn, J.P. (2001). Acid-stimulated phosphate uptake by activated sludge microorganisms under aerobic laboratory conditions.  Water Research, 35(18): 4317-4322.

8.  McGrath, J.W. & Quinn, J.P. (2000). Intracellular accumulation of polyphosphate by the yeast Candida humicola G-1 in response to acid pH.  Applied and Environmental Microbiology, 66: 4068-4073.

 

2. The biochemistry and genetics of organophosphate metabolism

Much of the effort of our laboratories has been devoted to obtaining a fuller understanding of the microbial metabolism of organic compounds that contain the metalloid elements arsenic, sulphur and phosphorus.

This has led, for example, to the discovery of novel routes for the cleavage of the carbon-arsenic bond (Quinn and McMullan: Microbiology (UK), 141: 721-727,1995) and the carbon-sulphur bond (King and Quinn: Microbiology (UK), 143: 3907-12, 1997; King et al.: Microbiology (UK), 143: 2339-43, 1997).  However our longest-running research theme has involved the study of the microbial metabolism of biogenic and xenobiotic organophosphonates - compounds that contain the highly-stable carbon - phosphorus bond.

Natural organophosphonates are widely distributed in biological materials either free or combined with lipids, polysaccharides or other cellular constituents.  Synthetic organophosphonates constitute a heterogenous group of molecules with diverse applications in industry and agriculture e.g. as herbicides, antibiotics, flame retardants, plasticizers, and lubricant - and detergent-additives.  Many thousands of tonnes of organophosphonates enter the environment each year: 70 million hectares of land are treated world-wide annually with the herbicide glyphosate (N-phosphonomethylglycine; trade names - Roundup, Clinic, Touchdown, Greenscape and Tumbleweed) while 10,000 tonnes of polyphosphonates are formulated within Europe per annum in household detergents.

Levels of both glyphosate application and phosphonate detergent usage are both expected to rise considerably within the next few years as glyphosate manufacturing costs fall, genetically resistant glyphosate crop plants are further developed and the use of phosphate in detergents declines.  Such widespread use has led to fears over the persistence of these C-P containing compounds in soils and natural waters: the persistence of contaminating organophosphonates in ground water has been extensively documented, while the DEFRA Drinking Water Inspectorate have recommended the development of monitoring strategies for glyphosate residues in water courses.  Concerns are further heightened by the extreme stability of the C-P bond to chemical, photolytic, and thermal decomposition and by the fact that many organophosphonates are powerful biocides.  Additionally the increased usage of organophosphonate detergent additives may also significantly increase the phosphorus load into sewage treatment works leading to the increased potential for the eutrophication of receiving waters.

Little detailed knowledge exists on the environmental fate of organophosphonates.  Various studies have reported only slow degradation during wastewater treatment and in natural waters.  No report of the isolation of microbial cultures able to mineralise even one of the organophosphonates presently used in detergents has ever been presented, while no microorganism capable of complete glyphosate mineralisation has yet been isolated (even though glyphosate degradation in soil ecosystems has been demonstrated).  This can be attributed to the fact that organophosphonate biodegradation has long been thought to be mediated by enzymes induced only under conditions of phosphate starvation i.e. under the control of the Pho or phosphate regulon: such conditions rarely pertain in the environment.  Organophosphonates therefore generally fail to serve as sources of carbon for microbial growth as the phosphate released during degradation serves to repress and/or inhibit further mineralisation.

Our work on organophosphonate degradation began as a collaboration with Zeneca plc on the metabolism of glyphosate by environmental microorganisms.  In subsequent BBSRC-funded studies [1992-1996 (Ref 81/P02873) and 1999-2002 (Ref 81/P11488)] we investigated the biochemical and genetic basis of microbial C-P bond cleavage in both xenobiotic and biogenic organophosphonates.  Our findings confirmed that the enzymes involved in the uptake and/or metabolism of such compounds were for the most part of the 'C-P lyase' type, and that their expression was regulated by a mechanism analogous to the classical Pho regulon.  Nevertheless we were able to establish that four other, novel, Pi-insensitive C-P bond cleavage enzymes exist within environmental microorganisms.  We identified:

1.  A previously-unreported C-P hydrolase specific to 2-oxo-1-hydroxypropyl phosphonic acid (in association with workers in the University of Vienna).  This is the key enzyme in a novel pathway for the degradation of the antibiotic phosphonomycin (Fig 1) (1,2-epoxy propylphosphonic acid) [McGrath et al.: Appl. Environ. Microbiol. 64: 356-58, 1998].


2.  A Pi-insensitive phosphonoacetaldehyde hydrolase in an environmental pseudomonad that utilizes 2-aminoethylphosphonic acid as a carbon source [Ternan and Quinn: Syst. Appl. Microbiol., 21: 346-52, 1998].

3.  Phosphonoacetate hydrolase [McMullan and Quinn: J. Bacteriol., 176: 320-324, 1994].  We purified and characterized this enzyme [McGrath et al.: Eur. J. Biochem., 234: 225-230, 1995]; determination of its X-ray crystal structure has now been carried out.  We also cloned the corresponding structural gene and in collaboration with a group in the Russian Academy of Sciences, Pushchino, showed that its induction is mediated by a new member of the LysR family of transcriptional regulators [Kulakova et al.: J. Bacteriol., 183: 3268-75, 2001].

4.  Phosphonopyruvate hydrolase (see Part 2, also Fig. 1) which produces pyruvate and Pi (Ternan and Quinn: Biochem. Biophys. Res. Commun., 248: 378-81, 1999). We have purified the enzyme and cloned its structural gene [Kulakova et al. J. Biol. Chem., 278: 23426-31, 2003; Chen et al. Biochemistry-US, 45: 11491-11504, 2006].

Further work on all aspects of the biochemistry and genetics of C-P bond metabolism by microorganisms, including the development of biosensors for important organophosphonate compounds is continuing, and we have recently published a review of the field [Quinn et. al. Environ. Microbiol., 9: 2392-2400, 2007].

Funding Obtained

McGrath, J.W. and Quinn, J.P. (2006). A novel biotechnological approach to phosphorus removal from wastewaters.  Environmental Protection Agency of Ireland €391494 (£274,000).

McGrath, J.W. and Quinn, J.P. (2005). Regulation of microbial carbon-phosphorus bond formation.  European Social Fund Studentship £40,000.

McGrath, J.W. (2004). In situ identification and isolation of process relevant microorganisms from a pilot plant employing a novel phosphate removal technology.  EPSRC  £123,060.

McGrath, J.W. (2004). In situ identification of activated sludge microorganisms which accumulate polyphosphate upon exposure to acid pH.  Royal Society  £14,550.

McGrath, J.W. (2003). Biodegradation of the organophosphonate phosphonomycin.  Nuffield Foundation New Lecturer Scheme.  £5,000.

McGrath, J.W. and Quinn, J.P. (2003). Sludge minimization - investigation of a novel approach.  QUESTOR Cast Award.  £31,100.